Centre for Plant Sciences

Prof Alison Baker

email: a.baker@leeds.ac.uk

Gene expression, Protein targeting and organelle biogenesis

The development and function of eukaryotic cells requires the accurate delivery of proteins to subcellular organelles. The consequence to the organism when these systems malfunction can be catastrophic. Our principal interest is in the biogenesis of peroxisomes and the targeting and import of peroxisomal proteins. In humans the failure of these mechanisms results in a debilitating and eventually fatal group of diseases, the peroxisome biogenesis disorders. In plants they are likely to have far ranging effects on seed viability, seedling vigour and the ability to withstand stress; all agronomically important traits. Our goal is to understand the molecular mechanism by which proteins enter peroxisomes and to understand how peroxisome biogenesis is regulated to meet the needs of the organism. We adopt a cross-disciplinary approach studying yeast, plant and mammalian cells as appropriate. We study the import of peroxisomal proteins and probe the interaction of translocation intermediates with the import machinery using an in vitro uptake system derived from sunflower cotyledons. This is complemented by in vivo targeting studies in yeast and plant cells. Expression of peroxisome biogenesis genes in plant and mammalian cells is studied using northerns and RT-PCR as well as promoter-luciferase gene fusions in transgenic plants. A new area of research is the isolation and characterisation of peroxisome mutants in the model plant Arabidopsis thaliana.

Schematic diagram of protein import into peroxisomes

Schematic diagram of protein import into peroxisomes

Most peroxisome matrix proteins possess one of two targeting signals, a C-terminal tripeptide called PTS1 or an N terminal nonapeptide termed PTS2. These proteins interact with the cytosolic receptors PEX5 and PEX7 respectively. In mammals and plants the two pathways are coupled, as an isoform of PEX5 (PEX5L) is required as an accessory protein for PEX7. In bakers yeast the two pathways are separate with PEX7 having 2 unique accessory proteins, PEX18 and PEX21. PEX5 and PEX7 receptors with their cargoes dock at a complex in the peroxisome membrane comprised of PEX14, 13 (and 17 in baker’s yeast). This docking complex is part of a larger complex ‘the importomer’ which includes the membrane proteins PEX2,10 and 12. By a mechanism that remains unknown, matrix proteins traverse the membrane, probably still associated with their receptor. PEX5 at least partially traverses the membrane and interacts with PEX8 on the trans side of the membrane. Subsequently cargo is unloaded and the receptors recycled, again by an unknown mechanism that involves PEX4 and 22. Insertion of peroxisome membrane proteins (PMPs) is less well understood but requires PEX19, which may function as a receptor/chaperone and PEX3. In mammals PEX16 is also involved in this process. (see Brown and Baker 2003 for further details).

Young Plant (20d) Young Plant (20d)
Senescent Plant (60d) Senescent Plant (60d)

PEX1 gene expression is up regulated in senescence.
Plants were transformed with luciferase under the control of the PEX1 promoter. In the young plant (grown 20 days in short day conditions) PEX1 expression as revealed by photon emission from the luciferase reporter gene is restricted to the youngest leaves and the apical meristem. In the same plant 40 days later which is visibly senescent (the lower leaves are yellow and wrinkled in appearance) high levels of photon emission are seen throughout the rosette.
 Mutants in the COMATOSE protein are defective in fatty acid mobilisation.
COMATOSE is an Arabidopsis peroxisome membrane protein belonging to the ABC transporter family. It shows homology to ALDP a human peroxisomal ABC transporter that is defective in the disease X-linked adrenoleukodystrophy. Comatose mutants are defective in mobilisation of seed triacylglycerol and green cotyledon cells from these mutants are packed with lipid bodies (top micrograph) that are absent from wild type cotyledon cells from otherwise comparable plants (bottom micrograph). Comatose mutants also show a range of other developmental and biochemical phenotypes (see Footitt et al., 2002 for further details).
 Arabidopsis peroxisomes (red) visualised with a PEX10-YFP fusion protein. Chloroplasts are shown in green.